Vectors with modified initiation codon for the translation of AAV-Rep78 useful for production of AAV
First Claim
1. A method for producing a recombinant parvoviral virion in an insect cell, comprising the steps of:
- (a) culturing an insect cell comprising;
(i) a first nucleotide sequence comprising;
(A) a single open reading frame (ORF) encoding parvoviral Rep proteins Rep78 and Rep52; and
(B) a suboptimal initiation codon selected from the group consisting of ACG, TTG, CTG and GTG, and(ii) not comprising a nucleotide sequence encoding parvoviral Rep proteins other than said first nucleotide sequence,under conditions such that the recombinant parvoviral virion is produced; and
(b) recovering the recombinant parvoviral virion.
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Abstract
The present invention relates nucleic acid constructs for the production of recombinant parvoviral (e.g. adeno-associated viral) vectors in insect cells, to insect cells comprising such constructs and to methods wherein the cells are used to produce recombinant parvoviral virions. The insect cells preferably comprise a first nucleotide sequence encoding the parvoviral rep proteins whereby the initiation codon for translation of the parvoviral Rep78 protein is a suboptimal initiation codon that effects partial exon skipping upon expression in insect cells. The insect cell further comprises a second nucleotide sequence comprising at least one parvoviral (AAV) inverted terminal repeat (ITR) nucleotide sequence and a third nucleotide sequence comprising a sequences coding for the parvoviral capsid proteins.
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Citations
21 Claims
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1. A method for producing a recombinant parvoviral virion in an insect cell, comprising the steps of:
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(a) culturing an insect cell comprising; (i) a first nucleotide sequence comprising; (A) a single open reading frame (ORF) encoding parvoviral Rep proteins Rep78 and Rep52; and (B) a suboptimal initiation codon selected from the group consisting of ACG, TTG, CTG and GTG, and (ii) not comprising a nucleotide sequence encoding parvoviral Rep proteins other than said first nucleotide sequence, under conditions such that the recombinant parvoviral virion is produced; and (b) recovering the recombinant parvoviral virion.
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2. The method according to claim 1, further comprising the step of affinity-purification of the virion using an anti-parvoviral antibody.
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3. The method according to claim 2, wherein the anti-parvoviral antibody is a single chain camelid antibody or a fragment thereof.
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4. The method according to claim 1, wherein the recombinant parvoviral virion is a recombinant adeno-associated virus (AAV) virion.
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5. The method according to claim 2, wherein the anti-parvoviral antibody is an immobilized antibody.
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6. The method according to claim 1, wherein the nucleotide sequence comprising the single ORF encoding the parvoviral Rep proteins is part of a nucleic acid construct wherein the ORF is operably linked to an expression control sequence for expression in said insect cells.
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7. The method according to claim 6 wherein the expression control sequence comprises a polyhedron promoter.
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8. The method according to claim 1, wherein the insect cell further comprises:
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(i) a second nucleotide sequence comprising at least one parvoviral inverted terminal repeat (ITR) sequence; and
,(ii) a third nucleotide sequence comprising parvoviral capsid protein-coding sequences operably linked to an expression control sequence for expression in the insect cell.
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9. The method according to claim 1, wherein the insect cell comprises a first and a second nucleic acid construct, wherein:
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(i) the first nucleic acid construct comprises; (A) the first nucleotide sequence of step (a), and (B) a third nucleic acid sequence that encodes a parvoviral capsid protein, both the first and the third nucleotide sequences being operably linked to an expression control sequence for expression in the insect cell; and
,(ii) the second nucleic acid construct comprising a second nucleotide sequence that includes at least one parvoviral ITR sequence.
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10. The method according to claim 9 wherein the first nucleic acid construct is an insect cell-compatible vector.
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11. The method according to claim 9, wherein the second nucleic acid construct is an insect cell-compatible vector.
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12. The method according to claim 8, wherein the second nucleotide sequence further comprises a nucleotide sequence encoding a gene product of interest wherein the nucleotide sequence encoding the gene product of interest becomes incorporated into the genome of the parvoviral vector produced in said cell.
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13. The method according to claim 12, wherein the second nucleotide sequence comprises two parvoviral ITR sequences which flank the nucleotide sequence encoding the gene product of interest.
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14. The method according to claim 8, wherein
the third nucleotide sequence comprising said capsid protein-coding sequences encodes parvoviral VP1, VP2, and VP3 capsid proteins, and the initiation codon for translation of the VP1 capsid protein is ACG, TTG, CTG, or GTG.
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15. The method according to claim 14, wherein the third nucleotide sequence comprises an expression control sequence comprising a nonanucleotide, the sequence of which is SEQ ID NO:
- 7, said control sequence being upstream of the initiation codon of the sequence encoding the VP1 protein.
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16. The method according to claim 14, wherein the third nucleotide sequence further comprises at least one of the following modifications of the parvoviral VP1-coding sequence, in which the position designated as position 1 is the 17th nucleotide of SEQ ID NO:
- 1;
a C at nucleotide position 12, an A at nucleotide position 21, or a C at nucleotide position 24.
- 1;
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17. The method according to claim 8, wherein at least one of
the first nucleotide sequence, the second nucleotide sequence, or the third nucleotide sequence is stably integrated in the genome of the insect cell.
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18. The method according to claim 1, wherein the parvoviral Rep proteins are AAV Rep proteins.
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19. The method according to claim 10, wherein the insect cell-compatible vector is a baculoviral vector.
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20. The method according to claim 11, wherein the insect cell-compatible vector is a baculoviral vector.
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21. The method according to claim 1, wherein the first nucleotide sequence comprises an expression control sequence comprising a nonanucleotide, the sequence of which is SEQ ID NO:
- 7, said control sequence being upstream of the initiation codon of the sequence encoding the Rep78 and Rep52 proteins.
Specification